Inhibiting Germination of Clostridium Perfringens Spores to Reduce Necrotic Enteritis

ABSTRACT

Provided herein are materials and methods useful for reducing, preventing, and/or inhibiting germination of  C. perfringens  spores, including methods for inhibiting  C. perfringens  germination to reduce necrotizing enteritis (NE, also referred to as necrotic enteritis) in animals. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/545,645, filed Jul. 21, 2017, which is a 35 U.S.C. § 371 U.S.National Stage of International Application No. PCT/US2016/016848, filedFeb. 5, 2016, which claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/113,184, filed on Feb. 6, 2015, thecontents of which are incorporated herein fully by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant no.2010-65119-20603, awarded by the United States Department ofAgriculture. The government has certain rights in the invention.

BACKGROUND

C. perfringens is a Gram-positive, rod-shaped, spore-forming, obligateanaerobic bacterium (Van Immerseel et al., Avian Pathol 2004,33(6):537-549; Shimizu et al., Proc Natl Acad Sci USA 2002,99(2):996-1001; Myers et al., Genome Res 2006, 16(8):1031-1040; andPetit et al., Trends Microbiol 1999, 7(3):104-110) that causes a widerange of diseases in humans and animals, ranging from food poisoning tosevere invasive disease (e.g., myonecrosis) (Van Immerseel et al.,Trends Microbiol 2009, 17(1):32-36). The ability of C. perfringens tocause disease is ascribed mainly to the differential production of fourmajor and ten minor protein toxins (Rood, Ann Rev Microbiol 1998,52(1):333-360; and Smedley et al., “The enteric toxins of Clostridiumperfringens,” In: Rev Physiol Biochem Pharmacol 2005:183-204).

In avian species, C. perfringens strains can cause necrotic enteritis(NE), which can result in substantial economic damage to commercialpoultry (Petit et al., supra). Chickens suffering from clinical NEappear depressed, anorexic, and relatively immobile (Broussard et al.,Avian Dis 1986, 30(3):617-619). Onset of disease is sudden, with deathensuing quickly (Wages and Opengart, “Necrotic enteritis,” in Diseasesof Poultry, 11th ed., Iowa State Press, Ames, Iowa, 2003). The acuteform of NE may cause up to 30% mortality in broiler flocks (Kaldhusdaland Lvland, World Poultry 2000a, 16:50-51; and Williams, Avian Pathol2005, 34(3):159-180). In the sub-clinical form, damage to the intestinalmucosa can lead to decreased digestion and absorption, reduced weightgain, and a poor feed conversion ratio (Elwinger et al., ActaVeterinaria Scandinavia 1992, 33:369-378; and Kaldhusdal et al., AvianDis 2001, 45(1):149-156).

C. perfringens spores are ubiquitous in the environment, andcolonization of poultry by C. perfringens occurs early in the life ofthe animals (Craven et al., Avian Dis 2003, 47(3):707-711; Craven etal., Avian Dis 2001, 45(4):887-896; and Barbara et al., VeterinaryMicrobiol 2008, 126(4):377-382). Most of these strains, however, arepart of the normal flora, and are incapable of initiating the diseaseprocess. Thus, the mere presence of C. perfringens in thegastrointestinal (GI) tract of broiler chickens is not sufficient forthe development of NE (Van Immerseel et al., supra; Kaldhusdal, WorldPoultry 2000b, 16(6):42-43; and Hermans and Morgan, Res Vet Science2003, 74(Suppl. 1):19). Rather, one or more predisposing factors may berequired to elicit the clinical signs and lesions of NE. For example, animportant predisposing factor in natural cases of NE may be intestinaldamage caused by coccidia (Williams, supra). These organisms can damageenterocytes, opening a pathway for the association of C. perfringenswith the mucosal epithelium. Coccidia also can alter the normal gutflora (Oviedo-Rondon et al., Poultry Sci 2006, 85(5): 854-860), whichcan allow for C. perfringens spore germination followed by colonizationof the empty intestinal niches by the vegetative, toxin-producing cells.Feed composition also is a potent risk factor. In particular, dietsbased on wheat or barley are far more likely to be associated withoutbreaks of NE than are diets based on corn (Kaldhusdal 2000b, supra;and Riddell and Kong, Avian Dis 1992, 36(3):499-503).

NE infections in the US often are controlled incidentally by in-feedantibiotic growth promoters (AGPs), which include well-knownantibacterial and antiparasitic drugs (Williams, supra). The phasing outof antibiotic growth promoters from poultry diets in Europe, however,has changed the microbial profile of the GI tract in commercial poultry(Yegani and Korver, Poultry Sci 2008, 87(10):2052-2063). In Scandinaviancountries, a ban on antimicrobial growth promoters was almostimmediately followed by an NE epidemic (Kaldhusdal 2000a, supra),leading to the use of greatly increased amounts of antimicrobials fortreatment. In addition, anti-coccidial compounds (mainly ionophores)have been removed from routine use due to the introduction of a highlyefficacious attenuated anti-coccidial vaccine (Crouch et al., AvianPathol 2003, 32(3):297-304). These ionophore compounds also areanti-clostridial, so their absence from feed can increase the incidenceof NE.

Due to the changes in commercial poultry feed supplementation, NE hasbecome a global economic problem (van der Sluis, World Poultry 2000,16(5):56-57). Moreover, methods for controlling NE in poultry are notwell established (Williams, supra), and most protocols rely on dietarymodifications to prevent NE (Riddell and Kong, supra). Although AGPs areeffective in clinical NE suppression (Elwinger et al., Acta VeterinariaScandinavia 1998, 39:433-441), prophylactic antibiotic use has beendiscouraged. Accordingly, there remains a need for methods of reducing,preventing, and/or treating NE in poultry. These needs and others aremet by the present invention.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied andbroadly described herein, the invention, in one aspect, relates tocompounds that can prevent germination of C. perfringens spores andmaterials and methods for reducing or preventing C. perfringens sporegermination, as well as materials and methods for reducing, preventing,or treating adverse effects associated with exposure to germinated C.perfringens, including NE.

Disclosed are methods for preventing a disease caused by infection by-Clostridium perfringens in a subject, the method comprisingadministering to the subject an effective amount of a compound having astructure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a) R^(5b) (C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided thatif Q is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino, thereby preventing the disease caused by infection byClostridium perfringens in a subject.

Also disclosed are methods for inhibiting germination of at least oneClostridium perfringens spore, the method comprising contacting thespore with a compound having a structure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino, thereby inhibiting germination of at least one Clostridiumperfringens spore.

Also disclosed are feed compositions comprising feed components and acompound having a structure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino.

Also disclosed are compositions containing a compound that, whenadministered to an animal, inhibits germination of Clostridiumperfringens spores in the gut of the animal. The compound can be acompound of Formula (I):

wherein X is selected from the group consisting of O, N, and S, and R¹and R² independently are selected from the group consisting of H, OH,CN, NO₂, halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.Alternatively, the compound can be MOB:

or a pharmaceutically acceptable salt thereof, or the compound can be2-MTB:

or a pharmaceutically acceptable salt thereof. The animal can be a farmanimal (e.g., a chicken, turkey, duck, goose, cow, sheep, horse, orpig). The composition can be formulated as feed for the animal.

Also disclosed are methods for inhibiting germination of a Clostridiumperfringens spore, comprising contacting the spore with a compositioncontaining MOB or a pharmaceutically acceptable salt thereof, 2-MTB or apharmaceutically acceptable salt thereof. The spore can be in the gut ofan animal. The animal can be a farm animal (e.g., a chicken, turkey,duck, goose, cow, sheep, horse, or pig).

Also disclosed are methods for reducing the occurrence of necrotizingenteritis in a population of animals, comprising administering to thepopulation a composition containing MOB or a pharmaceutically acceptablesalt thereof, or 2-MTB or a pharmaceutically acceptable salt thereof,wherein the compound is administered in an amount effective to preventgermination of Clostridium perfringens spores in the gut of at least onemember of the population. The population can be a population ofchickens, turkeys, ducks, geese, cattle, sheep, horses, or pigs. Themethod can include administering the compound via feed provided to thepopulation.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1 shows a representative diagram of a scheme for C. perfringensspore germination. Solid lines represent required co-germinants. Cappedlines represent germination inhibitors. Dashed lines representgermination enhancers.

FIG. 2A and FIG. 2B show representative data illustrating thatL-alanine/L-phenylalanine-mediated C. perfringens spore germination ispotentiated by L-arginine and inhibited by L-tryptophan. Specifically,FIG. 2A shows a representative graph plotting germination of C.perfringens spores in defined medium (open circles) or in a solutioncontaining 25 mM L-alanine, 5 mM L-phenylalanine and 50 mM NaHCO₃(filled circles), as followed by the decrease in optical density at 580nm (OD₅₈₀). For clarity, data are shown at 5 minute intervals. The datawere generated for spores from C. perfringens strain JGS 1936. Other C.perfringens strains yielded similar results. FIG. 2B shows arepresentative graph plotting relative germination rates for C.perfringens JGS 1936 spores treated with the indicated amino acidmixtures. Germination rates were calculated from the linear segment ofoptical density changes over time. Relative germination was calculatedas the fraction of the germination rate for spores treated withL-alanine/L-phenylalanine. Amino acids are represented by the one-lettercode. Error bars represent standard deviations of six independentmeasurements. * p<0.003 compared to L-alanine/L-phenylalanine.

FIG. 3A and FIG. 3B show representative data illustrating that C.perfringens spores germinate with bile salts and amino acids.Specifically, FIG. 3A shows a representative graph plotting relativegermination of C. perfringens JGS 1936 spores treated with taurocholateand the indicated individual amino acids. Relative germination wascalculated as the fraction of the germination rate for spores treatedwith L-alanine/taurocholate. Amino acids are represented by one-lettercode. Error bars represent standard deviations of six independentmeasurements. FIG. 3B shows a representative graph plotting relativegermination of C. perfringens JGS 1936 spores treated with L-alanine andindividual bile salts. Relative germination was calculated as thefraction of the germination rate for spores treated withL-alanine/taurocholate. Error bars represent standard deviations of sixindependent measurements.

FIG. 4A-D show representative data illustrating the effect of pH andions on C. perfringens spore germination. Specifically, FIG. 4A shows arepresentative graph plotting relative germination of C. perfringens JGS1936 spores treated with L-alanine and L-phenylalanine at different pHlevels in sodium phosphate buffer (open circles) or potassium phosphatebuffer (filled circles). Relative germination was calculated as thefraction of the germination rate for spores suspended in sodiumphosphate buffer, pH=6.5. Error bars represent standard deviations ofsix independent measurements. FIG. 4B shows a representative graphplotting relative germination of C. perfringens JGS 1936 spores treatedwith taurocholate and L-alanine at different pH levels in sodiumphosphate buffer (open circles) or potassium phosphate buffer (filledcircles). Relative germination was calculated as the fraction of thegermination rate for spores suspended in potassium phosphate buffer,pH=6.5. FIG. 4C shows a representative graph plotting relativegermination of C. perfringens JGS 1936 spores resuspended in eithersodium phosphate (white bars) or potassium phosphate (black bars).Samples were individually supplemented with KCl, KBr, NaCl, NaBr, KHCO₃,or NaHCO₃. Germination was initiated by addition of L-alanine andL-phenylalanine. Relative germination was calculated as the fraction ofthe germination rate for spores suspended in sodium phosphate buffer,pH=6.5. FIG. 4D shows a representative graph plotting relativegermination rate for C. perfringens JGS 1936 spores resuspended ineither sodium phosphate (white bars) or potassium phosphate (blackbars). Samples were individually supplemented with KCl, KBr, NaCl, NaBr,KHCO₃, or NaHCO₃. Germination was initiated by addition of taurocholateand L-alanine. Relative germination was calculated as the fraction ofthe germination rate for spores suspended in potassium phosphate buffer,pH=6.5.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon. Nothing herein is tobe construed as an admission that the present invention is not entitledto antedate such publication by virtue of prior invention. Further, thedates of publication provided herein may be different from the actualpublication dates, which can require independent confirmation.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate aspects, can also beprovided in combination in a single aspect. Conversely, various featuresof the disclosure which are, for brevity, described in the context of asingle aspect, can also be provided separately or in any suitablesubcombination.

For the terms “for example” and “such as,” and grammatical equivalentsthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. All measurements reported herein areunderstood to be modified by the term “about”, whether or not the termis explicitly used, unless explicitly stated otherwise.

The term “compound” as used herein is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted. Compounds herein identified by name or structure asone particular tautomeric form are intended to include other tautomericforms unless otherwise specified.

All compounds, and salts thereof (e.g., pharmaceutically acceptablesalts), can be found together with other substances such as water andsolvents (e.g., hydrates and solvates).

Compounds provided herein also can include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers that are isomeric protonation stateshaving the same empirical formula and total charge. Example prototropictautomers include ketone—enol pairs, amide—imidic acid pairs,lactam—lactim pairs, enamine—imine pairs, and annular forms where aproton can occupy two or more positions of a heterocyclic system, forexample, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be inequilibrium or sterically locked into one form by appropriatesubstitution.

Compounds provided herein can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include hydrogen, tritium, anddeuterium.

The phrase “pharmaceutically acceptable” is used herein to refer tothose compounds, materials, compositions, and/or dosage forms that are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

Also provided herein are pharmaceutically acceptable salts of thecompounds described herein. As used herein, the term “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the compounds provided herein include theconventional non-toxic salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. The pharmaceuticallyacceptable salts of the compounds provided herein can be synthesizedfrom the parent compound that contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two. In various aspects, anon-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol,ethanol, iso-propanol, or butanol) or acetonitrile (ACN) can be used.Lists of suitable salts are found in Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418and Journal of Pharmaceutical Science, 66, 2 (1977). Conventionalmethods for preparing salt forms are described, for example, in Handbookof Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH,2002.

In some embodiments, a compound provided herein, or salt thereof, issubstantially isolated. By “substantially isolated” is meant that thecompound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compound providedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds provided herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

As used herein, chemical structures that contain one or morestereocenters depicted with dashed and bold bonds (i.e.,

) are meant to indicate absolute stereochemistry of the stereocenter(s)present in the chemical structure. As used herein, bonds symbolized by asimple line do not indicate a stereo-preference. Unless otherwiseindicated to the contrary, chemical structures, which include one ormore stereocenters, illustrated herein without indicating absolute orrelative stereochemistry encompass all possible stereoisomeric forms ofthe compound (e.g., diastereomers and enantiomers) and mixtures thereof.Structures with a single bold or dashed line, and at least oneadditional simple line, encompass a single enantiomeric series of allpossible diastereomers.

Resolution of racemic mixtures of compounds can be carried out usingappropriate methods. An exemplary method includes fractionalrecrystallization using a chiral resolving acid that is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, for example, optically activeacids, such as the D and L forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, orthe various optically active camphorsulfonic acids such ascamphorsulfonic acid. Other resolving agents suitable for fractionalcrystallization methods include stereoisomerically pure forms ofmethylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent compositions canbe determined by one skilled in the art.

The expressions “ambient temperature” and “room temperature” as usedherein are understood in the art and refer generally to a temperature,e.g., a reaction temperature, that is about the temperature of the roomin which the reaction is carried out, for example, a temperature fromabout 20° C. to about 30° C.

At various places in the present specification, divalent linkingsubstituents are described. It is specifically intended that eachdivalent linking substituent include both the forward and backward formsof the linking substituent. For example, —NR(CR′R″)_(n)— includes both—NR(CR′R″)_(n)— and —(CR′R″)_(n)NR—. Where the structure clearlyrequires a linking group, the Markush variables listed for that groupare understood to be linking groups.

The term “n-membered” where n is an integer typically describes thenumber of ring-forming atoms in a moiety where the number ofring-forming atoms is n. For example, piperidinyl is an example of a6-membered heterocycloalkyl ring, pyrazolyl is an example of a5-membered heteroaryl ring, pyridyl is an example of a 6-memberedheteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a10-membered cycloalkyl group.

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. As used herein, the term “substituted” means that ahydrogen atom is removed and replaced by a substituent. It is to beunderstood that substitution at a given atom is limited by valency.

The term “alkyl” includes substituted or unsubstituted straight-chainalkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, etc.) and branched-chain alkyl groups (isopropyl,tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has sixor fewer carbon atoms in its backbone (e.g., C₁₋₆ for straight chain;C₃₋₆ for branched chain). The term C₁₋₆ includes alkyl groups containing1 to 6 carbon atoms. In certain embodiments, a straight chain alkyl hasthree or fewer carbon atoms in its backbone. The term C₁₋₃ includesalkyl groups containing one to three carbon atoms.

As used herein, “haloalkyl” means a hydrocarbon substituent, which is alinear or branched or cyclic alkyl, alkenyl or alkynyl substituted withone or more chloro, bromo, fluoro, or iodo atom(s). In some embodiments,a haloalkyl is a fluoroalkyl, wherein one or more of the hydrogen atomshave been substituted by fluoro. In some embodiments, haloalkyls are oneto about three carbons in length (e.g., one to about two carbons inlength, or one carbon in length).

The term “alkoxy” includes groups of the formula —OR, where R is analkyl as defined herein. Non-limiting examples of alkoxy groups includemethoxy, ethoxy, isopropoxy, tert-butoxy, and the like. In someembodiments, an alkoxy group can have from one to three carbons (e.g.,methyoxy, ethoxy, or propoxy).

The term “haloalkoxy” includes group of the formula —OR, where R is ahaloalkyl as defined herein. Examples of haloalkoxy groups include,without limitation, trifluoromethoxy, difluoromethoxy, etc.

“Alkylamino” includes groups of the formula —NR, where R is an alkyl asdefined herein. Non-limiting examples of alkylamino groups includemethylamino, ethylamino, isopropylamino, butylamino etc. In someembodiments, an alkylamino group can have from one to three carbons(e.g., methyoxy, ethoxy, or propoxy). The term “dialkylamino” includesgroups of the formula —NR₂, where R is an alkyl as defined herein. Insome embodiments, the alkyl groups of a dialkylamino independently canhave one to three carbons.

In general, the term “aryl” includes substituted or unsubstitutedaromatic rings, including 5- and 6-membered single-ring aromatic groups,such as benzene and phenyl. Further, the term “aryl” includesmulticyclic aryl groups, e.g., tricyclic, bicyclic, such as naphthaleneand anthracene. In some embodiments, aryls can have from six to ten(e.g., six, seven, eight, nine, or ten) ring atoms.

The term “heteroaryl” means a substituted or unsubstituted mono-, bi-,tri- or polycyclic group having four to 14 ring atoms, alternativelyfive, six, nine, or ten ring atoms; having six, ten, or 14 pi electronsshared in a cyclic array; wherein at least one ring in the system isaromatic, and at least one ring in the system contains one or moreheteroatoms independently selected from the group consisting of N, O,and S. Exemplary heteroaryl groups include, for example, pyrrole, furan,thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole,pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like. Further, the term “heteroaryl” includesmulticyclic heteroaryl groups, e.g., tricyclic or bicyclic groups, suchas benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,napthyridine, indole, benzofuran, purine, benzofuran, quinazoline,deazapurine, indazole, or indolizine.

The term “heterocycloalkyl” includes substituted or unsubstitutedgroups, including but not limited to, three- to ten-membered single ormultiple rings having one to five heteroatoms, for example, piperazine,pyrrolidine, piperidine, or homopiperazine. In certain embodiments, aheterocycloalkyl can have from four to ten ring atoms.

Methods for making compounds as described herein include those known inthe art; such compounds also may be obtained commercially (e.g., fromSigma-Aldrich, St. Louis, Mo.). In some embodiments, benzoazolederivatives can be generated by modifying the benzyl ring, azole ring,and/or side chain of MOB or 2-MTB, as indicated in Formula (I).Derivatives can include, for example, various benzoimidazoles (whereX═N), benzoxazoles (where X═O), and benzothiazoles (where X═S). Thederivative compounds can be tested for anti-germination activity, andthen tested as NE prophylactics. Information gathered from such in vitroand in vivo screens can be used to direct further derivatization.

The term “substituted” means that an atom or group of atoms replaceshydrogen as a “substituent” attached to another group. For aryl andheteroaryl groups, the term “substituted”, unless otherwise indicated,refers to any level of substitution, namely mono, di, tri, tetra, orpenta substitution, where such substitution is permitted. Thesubstituents are independently selected, and substitution may be at anychemically accessible position. In some cases, two sites of substitutionmay come together to form a 3-10 membered cycloalkyl or heterocycloalkylring. Non-limiting examples of substituents include: (C₁-C₆)alkyl, halo,(C₁-C₆)haloalkyl, —CN, —NR⁸R⁹, —NO₂, —O(C₁-C₆)haloalkyl, —OR⁸, —OC(O)R⁸,—C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R⁹, —SR⁸, —S(O)R⁸, —SO₂R⁸, —SO₂NR⁸R⁹, (C₃-C₇)cycloalkyl, (C₃-C₇)heterocycloalkyl, (C₅-C₁₄)aryl, and(C₅-C₁₄)heteroaryl, wherein R⁸ and R⁹ are independently selected from Hand (C₁-C₆)alkyl.

It is to be noted that this document encompasses not only the variousisomers of the compounds that may exist, but also the various mixturesof isomers that may be formed, as well as any enantiomers and tautomersthat may exist.

In addition, the scope of this document also encompasses solvates andsalts of the compounds described herein, as well as prodrugs of thecompounds, such as esters, amides, and acylated groups, among others. Insome embodiments, for example, this document provides prodrugs of thecompounds disclosed herein, which may contain, for example, acylatedphenols or acyl derivatives of amines. By “prodrug” is meant, forexample, any compound (whether itself active or inactive) that isconverted chemically in vivo into a biologically active compound asprovided herein, following administration of the prodrug to a subject.In some embodiments, a prodrug is a covalently bonded carrier thatreleases the active parent drug when administered to a subject. Prodrugscan be prepared by modifying functional groups present in the compoundsin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compounds. Prodrugs can includecompounds in which hydroxyl, amino, sulfhydryl, or carboxyl groups arebonded to any group that, when administered to a mammalian subject,cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl grouprespectively. Examples of prodrugs include, without limitation, acetate,formate and benzoate derivatives of alcohol and amine functional groupsin the compounds provided herein. The suitability and techniquesinvolved in making and using prodrugs are discussed in Higuchi andStella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the ACSSymposium Series, and in Bioreversible Carriers in Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987, both of which are hereby incorporated by reference in theirentirety.

The phrase “pharmaceutically acceptable” is used herein to refer tothose compounds, materials, compositions, and/or dosage forms that are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. Examples of pharmaceutically acceptablesalts of the compounds provided herein include acid addition salts andbase salts of the compounds.

B. Compounds

In one aspect, the invention relates to compounds useful in preventingdiseases associated with infection caused by C. perfringens, inparticular necrotizing enteritis. Thus, this document provides compoundsfor preventing, treating, or reducing NE in fowl and other farmedanimals. The compounds can be, for example, MOB and 2-MTB, as shownbelow.

-   -   (MOB) (2-MTB)

Alternatively, the compounds can be, for example, a benzoazole compoundthat is a derivative of MOB and/or 2-MTB. Such derivatives can include,without limitation, derivatization of the benzyl ring, the S in theazole ring, and/or the carbon between the S and N within the azole ring.Thus, in various aspects, a compound can be a compound of Formula (I):

where X can be selected from the group consisting of O, N, and S, and R¹and R² independently can be selected from the group consisting of H, OH,CN, NO₂, halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In one aspect, the disclosed compounds exhibit inhibition of germinationof C. perfringens spores.

In one aspect, the compounds of the invention are useful in theprevention of diseases associated with infection caused by C.perfringens, as further described herein.

It is contemplated that each disclosed derivative can be optionallyfurther substituted. It is also contemplated that any one or morederivative can be optionally omitted from the invention. It isunderstood that a disclosed compound can be provided by the disclosedmethods. It is also understood that the disclosed compounds can beemployed in the disclosed methods of using.

1. Structure

In one aspect, disclosed are compounds having a structure represented bya formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino.

In a further aspect, Z is selected from O and NR⁴; wherein R¹ isselected from hydrogen, C1-C3 alkyl, and C1-C3 haloalkyl; wherein eachof R^(2a), R^(2b), R^(2c), and R^(2d) is independently selected fromhydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C3 alkyl, C1-C3 alkoxy,C1-C3 haloalkyl, C1-C3 alkylamino, and (C1-C3)(C1-C3) dialkylamino; andwherein R⁴, when present, is selected from hydrogen and C1-C3 alkyl.

In a further aspect, the compound has a structure represented by aformula selected from:

In a further aspect, the compound has a structure represented by aformula selected from:

In a further aspect, the compound has a structure represented by aformula selected from:

In a further aspect, the compound has a structure represented by aformula:

In a further aspect, the compound is selected from:

a. Q Groups

In one aspect, Q is selected from O, S, and NR³. In a further aspect, Qis selected from O and NR³. In a still further aspect, Q is selectedfrom S and NR³. In yet a further aspect, Q is selected from O and S. Inan even further aspect, Q is O. In a still further aspect, Q is S. Inyet a further aspect, Q is NR³.

b. Z Groups

In one aspect, Z is selected from O, S, and NR⁴. In a further aspect, Zis selected from O and NR⁴. In a still further aspect, Z is selectedfrom S and NR⁴. In yet a further aspect, Z is selected from O and S. Inan even further aspect, Z is O. In a still further aspect, Z is S. Inyet a further aspect, Z is NR⁴.

c. R¹ Groups

In one aspect, R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵. In a further aspect, R¹is selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl,—CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar². In a still further aspect,R¹ is hydrogen.

In a further aspect, R¹ is selected from —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹,Cy¹, and Ar². In a still further aspect, R¹ is selected from Cy¹ andAr². In yet a further aspect, R¹ is Cy¹. In a still further aspect, R¹is Ar². In yet a further aspect, R¹ is —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹.

In a further aspect, R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, Cy¹, and Ar². In a still further aspect, R¹ is selected fromhydrogen, C1-C4 alkyl, C1-C4 haloalkyl, Cy¹, and Ar². In yet a furtheraspect, R¹ is selected from hydrogen, methyl, ethyl, n-propyl, i-propyl,—CH₂F, —CH₂Cl, —CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br, —(CH₂)₂CH₂F,—(CH₂)₂CH₂Cl, —(CH₂)₂CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃, —CHBr₂,—CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂, —CH₂CCl₃, —CH₂CHBr₂, —CH₂CBr₃,—(CH₂)₂CHF₂, —(CH₂)₂CF₃, —(CH₂)₂CHCl₂, —(CH₂)₂CCl₃, —(CH₂)₂CHBr₂,—(CH₂)₂CBr₃, Cy¹, and Ar². In an even further aspect, R¹ is selectedfrom hydrogen, methyl, ethyl, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂CH₂F,—CH₂CH₂Cl, —CH₂CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃, —CHBr₂, —CBr₃,—CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂, —CH₂CCl₃, —CH₂CHBr₂, —CH₂CBr₃, Cy¹, andAr². In a still further aspect, R¹ is selected from hydrogen, methyl,—CH₂F, —CH₂Cl, —CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃, —CHBr₂, Cy¹, and Ar².

In a further aspect, R¹ is selected from hydrogen, C1-C8 alkyl, andC1-C8 haloalkyl. In a still further aspect, R¹ is selected fromhydrogen, C1-C4 alkyl, and C1-C4 haloalkyl. In yet a further aspect, R¹is selected from hydrogen, methyl, ethyl, n-propyl, i-propyl, —CH₂F,—CH₂Cl, —CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br, —(CH₂)₂CH₂F,—(CH₂)₂CH₂Cl, —(CH₂)₂CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃, —CHBr₂, —CBr₃,—CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂, —CH₂CCl₃, —CH₂CHBr₂, —CH₂CBr₃,—(CH₂)₂CHF₂, —(CH₂)₂CF₃, —(CH₂)₂CHCl₂, —(CH₂)₂CCl₃, —(CH₂)₂CHBr₂, and—(CH₂)₂CBr₃. In an even further aspect, R¹ is selected from hydrogen,methyl, ethyl, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br,—CHF₂, —CF₃, —CHCl₂, —CCl₃, —CHBr₂, —CBr₃, —CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂,—CH₂CCl₃, —CH₂CHBr₂, and —CH₂CBr₃. In a still further aspect, R¹ isselected from hydrogen, methyl, —CH₂F, —CH₂Cl, —CH₂Br, —CHF₂, —CF₃,—CHCl₂, —CCl₃, —CHBr₂, and —CBr₃.

In a further aspect, R¹ is selected from hydrogen and C1-C8 haloalkyl.In a still further aspect, R¹ is selected from hydrogen and C1-C4haloalkyl. In yet a further aspect, R¹ is selected from hydrogen, —CH₂F,—CH₂Cl, —CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br, —(CH₂)₂CH₂F,—(CH₂)₂CH₂Cl, —(CH₂)₂CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃, —CHBr₂, —CBr₃,—CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂, —CH₂CCl₃, —CH₂CHBr₂, —CH₂CBr₃,—(CH₂)₂CHF₂, —(CH₂)₂CF₃, —(CH₂)₂CHCl₂, —(CH₂)₂CCl₃, —(CH₂)₂CHBr₂, and—(CH₂)₂CBr₃. In an even further aspect, R¹ is selected from hydrogen,—CH₂F, —CH₂Cl, —CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br, —CHF₂, —CF₃,—CHCl₂, —CCl₃, —CHBr₂, —CBr₃, —CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂, —CH₂CCl₃,—CH₂CHBr₂, and —CH₂CBr₃. In a still further aspect, R¹ is selected fromhydrogen, —CH₂F, —CH₂Cl, —CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃, —CHBr₂, and—CBr₃.

In a further aspect, R¹ is selected from hydrogen and C1-C8 alkyl. In astill further aspect, R¹ is selected from hydrogen and C1-C4 alkyl. Inyet a further aspect, R¹ is selected from hydrogen, methyl, ethyl,n-propyl, and i-propyl. In an even further aspect, R¹ is selected fromhydrogen, methyl, and ethyl. In a still further aspect, R¹ is selectedfrom hydrogen and ethyl. In yet a further aspect, R¹ is selected fromhydrogen and methyl.

In a further aspect, R¹ is C1-C8 alkyl. In a still further aspect, R¹ isC1-C4 alkyl. In yet a further aspect, R¹ is selected from methyl, ethyl,n-propyl, and i-propyl. In an even further aspect, R¹ is selected frommethyl and ethyl. In a still further aspect, R¹ is ethyl. In yet afurther aspect, R¹ is methyl.

d. R^(2A), R^(2B), R^(2C), and R^(2D) Groups

In one aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂,C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In a further aspect, each of R^(2a),R^(2b), R^(2c), and R^(2d) is hydrogen.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, —F, —Cl, —Br, —OH, —CN, —NO₂,—NH₂, methyl, ethyl, n-propyl, i-propyl, —OCH₃, —OCH₂CH₃, —O(CH₂)₂CH₃,—OCH(CH₃)₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br,—(CH₂)₂CH₂F, —(CH₂)₂CH₂Cl, —(CH₂)₂CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃,—CHBr₂, —CBr₃, —CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂, —CH₂CCl₃, —CH₂CHBr₂,—CH₂CBr₃, —(CH₂)₂CHF₂, —(CH₂)₂CF₃, —(CH₂)₂CHCl₂, —(CH₂)₂CCl₃,—(CH₂)₂CHBr₂, —(CH₂)₂CBr₃, —NHCH₃, —NHCH₂CH₃, —NH(CH₂)₂CH₃, —NHCH(CH₃)₂,—N(CH₃)₂, —N(CH₂CH₃)₂, —N((CH₂)₂CH₃)₂, —N(CH(CH₃)₂)₂, —N(CH₃)CH₂CH₃,—N(CH₃)(CH₂)₂CH₃, and —N(CH₃)CH(CH₃)₂. In a still further aspect, eachof R^(2a), R^(2b), R^(2c), and R^(2d) is independently selected fromhydrogen, —F, —Cl, —Br, —OH, —CN, —NO₂, —NH₂, methyl, ethyl, —OCH₃,—OCH₂CH₃, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br, —CHF₂,—CF₃, —CHCl₂, —CCl₃, —CHBr₂, —CBr₃, —CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂,—CH₂CCl₃, —CH₂CHBr₂, —CH₂CBr₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —N(CH₂CH₃)₂,and —N(CH₃)CH₂CH₃. In yet a further aspect, each of R^(2a), R^(2b),R^(2c), and R^(2d) is independently selected from hydrogen, —F, —Cl,—Br, —OH, —CN, —NO₂, —NH₂, methyl, —OCH₃, —CH₂F, —CH₂Cl, —CH₂Br, —CHF₂,—CF₃, —CHCl₂, —CCl₃, —CHBr₂, —CBr₃, —NHCH₃, and —N(CH₃)₂.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, C1-C4 alkyl, C1-C4alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In a still further aspect, each of R^(2a), R^(2b), R^(2c),and R^(2d) is independently selected from hydrogen, —F, —Cl, —Br,methyl, ethyl, n-propyl, i-propyl, —OCH₃, —OCH₂CH₃, —O(CH₂)₂CH₃,—OCH(CH₃)₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br,—(CH₂)₂CH₂F, —(CH₂)₂CH₂Cl, —(CH₂)₂CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃,—CHBr₂, —CBr₃, —CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂, —CH₂CCl₃, —CH₂CHBr₂,—CH₂CBr₃, —(CH₂)₂CHF₂, —(CH₂)₂CF₃, —(CH₂)₂CHCl₂, —(CH₂)₂CCl₃,—(CH₂)₂CHBr₂, —(CH₂)₂CBr₃, —NHCH₃, —NHCH₂CH₃, —NH(CH₂)₂CH₃, —NHCH(CH₃)₂,—N(CH₃)₂, —N(CH₂CH₃)₂, —N((CH₂)₂CH₃)₂, —N(CH(CH₃)₂)₂, —N(CH₃)CH₂CH₃,—N(CH₃)(CH₂)₂CH₃, and —N(CH₃)CH(CH₃)₂. In yet a further aspect, each ofR^(2a), R^(2b), R^(2c), and R^(2d) is independently selected fromhydrogen, —F, —Cl, —Br, methyl, ethyl, —OCH₃, —OCH₂CH₃, —CH₂F, —CH₂Cl,—CH₂Br, —CH₂CH₂F, —CH₂CH₂Cl, —CH₂CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃,—CHBr₂, —CBr₃, —CH₂CHF₂, —CH₂CF₃, —CH₂CHCl₂, —CH₂CCl₃, —CH₂CHBr₂,—CH₂CBr₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —N(CH₂CH₃)₂, and —N(CH₃)CH₂CH₃.In an even further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, —F, —Cl, —Br, methyl, —OCH₃,—CH₂F, —CH₂Cl, —CH₂Br, —CHF₂, —CF₃, —CHCl₂, —CCl₃, —CHBr₂, —CBr₃,—NHCH₃, and —N(CH₃)₂.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen and halogen. In a still furtheraspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, —F, —Cl, and —Br. In yet a further aspect, eachof R^(2a), R^(2b), R^(2c), and R^(2d) is independently selected fromhydrogen, —F, and —Cl. In an even further aspect, each of R^(2a),R^(2b), R^(2c), and R^(2d) is independently selected from hydrogen and—Cl. In a still further aspect, each of R^(2a), R^(2b), R^(2c), andR^(2d) is independently selected from hydrogen and —F.

In a further aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen and C1-C4 alkyl. In a still furtheraspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, methyl, ethyl, n-propyl, and i-propyl. In yet afurther aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, methyl, and ethyl. In an evenfurther aspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen and ethyl. In a still furtheraspect, each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen and methyl.

e. R³ Groups

In one aspect, R³, when present, is selected from hydrogen and C1-C8alkyl. In a further aspect, R³, when present, is selected from hydrogenand C1-C4 alkyl. In a still further aspect, R³, when present, ishydrogen.

In a further aspect, R³, when present, is selected from methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, and t-butyl. In a stillfurther aspect, R³, when present, is selected from methyl, ethyl,n-propyl, and i-propyl. In yet a further aspect, R³, when present, isselected from methyl and ethyl. In an even further aspect, R³, whenpresent, is ethyl. In a still further aspect, R³, when present, ismethyl.

f. R⁴ Groups

In one aspect, R⁴, when present, is selected from hydrogen and C1-C8alkyl. In a further aspect, R⁴, when present, is selected from hydrogenand C1-C4 alkyl. In a still further aspect, R⁴, when present, ishydrogen.

In a further aspect, R⁴, when present, is selected from methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, and t-butyl. In a stillfurther aspect, R⁴, when present, is selected from methyl, ethyl,n-propyl, and i-propyl. In yet a further aspect, R⁴, when present, isselected from methyl and ethyl. In an even further aspect, R⁴, whenpresent, is ethyl. In a still further aspect, R⁴, when present, ismethyl.

g. R^(S)A And R^(5B) Groups

In one aspect, each of R^(5a) and R^(5b), when present, is independentlyselected from hydrogen and C1-C4 alkyl. In a further aspect, each ofR^(5a) and R^(5b), when present, is hydrogen.

In a further aspect, each of R^(5a) and R^(5b), when present, isindependently selected from hydrogen, methyl, ethyl, n-propyl, andi-propyl. In a still further aspect, each of R^(5a) and R^(5b), whenpresent, is independently selected from hydrogen, methyl, and ethyl. Inyet a further aspect, each of R^(5a) and R^(5b), when present, isindependently selected from hydrogen and ethyl. In an even furtheraspect, each of R^(5a) and R^(5b), when present, is independentlyselected from hydrogen and methyl.

In a further aspect, each of R^(5a) and R^(5b), when present, isindependently selected from methyl, ethyl, n-propyl, i-propyl, n-butyl,i-butyl, s-butyl, and t-butyl. In a still further aspect, each of R^(5a)and R^(5b), when present, is independently selected from methyl, ethyl,n-propyl, and i-propyl. In yet a further aspect, each of R^(5a) andR^(5b), when present, is independently selected from methyl and ethyl.In an even further aspect, each of R^(5a) and R^(5b), when present, isethyl. In a still further aspect, each of R^(5a) and R^(5b), whenpresent, is methyl.

h. R⁶ Groups

In one aspect, R⁶, when present, is selected from hydrogen and C1-C4alkyl. In a further aspect, R⁶, when present, is hydrogen.

In a further aspect, R⁶, when present, is selected from hydrogen,methyl, ethyl, n-propyl, and i-propyl. In a still further aspect, R⁶,when present, is selected from hydrogen, methyl, and ethyl. In yet afurther aspect, R⁶, when present, is selected from hydrogen and ethyl.In an even further aspect, R⁶, when present, is selected from hydrogenand methyl.

In a further aspect, R⁶, when present, is selected from methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, and t-butyl. In a stillfurther aspect, R⁶, when present, is from methyl, ethyl, n-propyl, andi-propyl. In yet a further aspect, R⁶, when present, is from methyl andethyl. In an even further aspect, R⁶, when present, is ethyl. In a stillfurther aspect, R⁶, when present, is methyl.

i. AR¹ Groups

In one aspect, Ar¹ is selected from aryl and heteroaryl and substitutedwith 0, 1, or 2 groups independently selected from halogen, —OH, —CN,—NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a further aspect, Ar¹ isselected from aryl and heteroaryl and substituted with 0 or 1 groupselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In astill further aspect, Ar¹ is selected from aryl and heteroaryl andmonosubstituted with a group selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In yet a further aspect, Ar¹ is selectedfrom aryl and heteroaryl and unsubstituted.

In a further aspect, Ar¹ is aryl substituted with 0, 1, or 2 groupsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In a still further aspect, Ar¹ is aryl substituted with 0or 1 group selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In yet a further aspect, Ar¹ is aryl monosubstituted witha group selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In an even further aspect, A¹ is unsubstituted aryl.

In a further aspect, Ar¹ is phenyl substituted with 0, 1, or 2 groupsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In a still further aspect, Ar¹ is phenyl substituted with0 or 1 group selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In yet a further aspect, Ar¹ is phenyl monosubstitutedwith a group selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In an even further aspect, A¹ is unsubstituted phenyl.

In a further aspect, Ar¹ is heteroaryl substituted with 0, 1, or 2groups independently selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In a still further aspect, Ar¹ isheteroaryl substituted with 0 or 1 group selected from halogen, —OH,—CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect, A¹is heteroaryl monosubstituted with a group selected from halogen, —OH,—CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In an even further aspect,Ar¹ is unsubstituted heteroaryl.

In a further aspect, Ar¹ is selected from triazolyl, imidazolyl,pyrazolyl, pyrrolyl, benzothiophenyl, benzofuranyl, furanyl, thiophenyl,pyridinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl, and purinyland substituted with 0, 1, or 2 groups independently selected fromhalogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a stillfurther aspect, Ar¹ is selected from triazolyl, imidazolyl, pyrazolyl,pyrrolyl, benzothiophenyl, benzofuranyl, furanyl, thiophenyl, pyridinyl,pyrimidinyl, indolyl, quinolinyl, isoquinolinyl, and purinyl andsubstituted with 0 or 1 group selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In yet a further aspect, Ar¹ is selectedfrom triazolyl, imidazolyl, pyrazolyl, pyrrolyl, benzothiophenyl,benzofuranyl, furanyl, thiophenyl, pyridinyl, pyrimidinyl, indolyl,quinolinyl, isoquinolinyl, and purinyl and monosubstituted with a groupselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. Inan even further aspect, A¹ is selected from triazolyl, imidazolyl,pyrazolyl, pyrrolyl, benzothiophenyl, benzofuranyl, furanyl, thiophenyl,pyridinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl, and purinyland unsubstituted.

j. AR² Groups

In one aspect, Ar² is selected from aryl and heteroaryl and substitutedwith 0, 1, or 2 groups independently selected from halogen, —OH, —CN,—NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a further aspect, Ar² isselected from aryl and heteroaryl and substituted with 0 or 1 groupselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In astill further aspect, Ar² is selected from aryl and heteroaryl andmonosubstituted with a group selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In yet a further aspect, Ar² is selectedfrom aryl and heteroaryl and unsubstituted.

In a further aspect, Ar² is aryl substituted with 0, 1, or 2 groupsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In a still further aspect, Ar² is aryl substituted with 0or 1 group selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In yet a further aspect, Ar² is aryl monosubstituted witha group selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In an even further aspect, Ar² is unsubstituted aryl.

In a further aspect, Ar² is phenyl substituted with 0, 1, or 2 groupsindependently selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In a still further aspect, Ar² is phenyl substituted with0 or 1 group selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In yet a further aspect, Ar² is phenyl monosubstitutedwith a group selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In an even further aspect, Ar² is unsubstituted phenyl.

In a further aspect, Ar² is heteroaryl substituted with 0, 1, or 2groups independently selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In a still further aspect, Ar² isheteroaryl substituted with 0 or 1 group selected from halogen, —OH,—CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect,Ar² is heteroaryl monosubstituted with a group selected from halogen,—OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In an even further aspect,Ar² is unsubstituted heteroaryl.

In a further aspect, Ar² is selected from triazolyl, imidazolyl,pyrazolyl, pyrrolyl, benzothiophenyl, benzofuranyl, furanyl, thiophenyl,pyridinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl, and purinyland substituted with 0, 1, or 2 groups independently selected fromhalogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a stillfurther aspect, Ar² is selected from triazolyl, imidazolyl, pyrazolyl,pyrrolyl, benzothiophenyl, benzofuranyl, furanyl, thiophenyl, pyridinyl,pyrimidinyl, indolyl, quinolinyl, isoquinolinyl, and purinyl andsubstituted with 0 or 1 group selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In yet a further aspect, Ar² is selectedfrom triazolyl, imidazolyl, pyrazolyl, pyrrolyl, benzothiophenyl,benzofuranyl, furanyl, thiophenyl, pyridinyl, pyrimidinyl, indolyl,quinolinyl, isoquinolinyl, and purinyl and and monosubstituted with agroup selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino. In an even further aspect, Ar² is selected from triazolyl,imidazolyl, pyrazolyl, pyrrolyl, benzothiophenyl, benzofuranyl, furanyl,thiophenyl, pyridinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl,and purinyl and unsubstituted.

k. CV Groups

In one aspect, Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In afurther aspect, Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0 or 1 group selected fromhalogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a stillfurther aspect, Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and monosubstituted with a group selected from halogen,—OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect,Cy¹ is selected from C3-C7 cycloalkyl and C2-C7 heterocycloalkyl andunsubstituted.

In a further aspect, Cy¹ is C3-C7 cycloalkyl substituted with 0, 1, or 2groups independently selected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In a still further aspect, Cy¹ is C3-C7cycloalkyl substituted with 0 or 1 group selected from halogen, —OH,—CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect,Cy¹ is C3-C7 cycloalkyl monosubstituted with a group selected fromhalogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In an evenfurther aspect, Cy¹ is unsubstituted C3-C7 cycloalkyl.

In a further aspect, Cy¹ is selected from cyclopropyl, cyclopentyl, andcyclohexyl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a stillfurther aspect, Cy¹ is selected from cyclopropyl, cyclopentyl, andcyclohexyl and substituted with 0 or 1 group selected from halogen, —OH,—CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect,Cy¹ is selected from cyclopropyl, cyclopentyl, and cyclohexyl andmonosubstituted with a group selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In an even further aspect, Cy¹ is selectedfrom cyclopropyl, cyclopentyl, and cyclohexyl and unsubstituted.

In a further aspect, Cy¹ is C2-C7 heterocycloalkyl substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In a still further aspect, Cy¹ is C2-C7heterocycloalkyl substituted with 0 or 1 group selected from halogen,—OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect,Cy¹ is C2-C7 heterocycloalkyl monosubstituted with a group selected fromhalogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In an evenfurther aspect, Cy¹ is unsubstituted C2-C7 heterocycloalkyl.

In a further aspect, Cy¹ is selected from pyrrolidinyl,tetrahydrothiophenyl, furanyl, piperidinyl, and tetrahydropyranyl andsubstituted with 0, 1, or 2 groups independently selected from halogen,—OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a still further aspect,Cy¹ is selected from pyrrolidinyl, tetrahydrothiophenyl, furanyl,piperidinyl, and tetrahydropyranyl and substituted with 0 or 1 groupselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. Inyet a further aspect, Cy¹ is selected from pyrrolidinyl,tetrahydrothiophenyl, furanyl, piperidinyl, and tetrahydropyranyl andmonosubstituted with a group selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino. In an even further aspect, Cy¹ is selectedfrom pyrrolidinyl, tetrahydrothiophenyl, furanyl, piperidinyl, andtetrahydropyranyl and unsubstituted.

2. Example Compounds

In one aspect, a compound can be present as one or more of the followingstructures:

3. Prophetic Compound Examples

The following compound examples are prophetic, and can be prepared usingthe synthesis methods described herein above and other general methodsas needed as would be known to one skilled in the art. It is anticipatedthat the prophetic compounds would be active as inhibitors ofgermination of C. perfringens spores, and such activity can bedetermined using the assay methods described herein.

In one aspect, a compound can be selected from:

or a pharmaceutically acceptable salt thereof.

C. C. Perfringens

The first step in C. perfringens pathogenesis is the germination ofingested spores into replicating bacteria in the gut of hosts. Thus, asdescribed herein, it is possible that compounds able to curtail C.perfringens spore germination will also prevent NE. For example,anti-germinants may be added as supplements to the feed of farmed fowl(e.g., chickens, turkeys, geese, and ducks), as well as to the feed ofother farm animals such as cattle, sheep, horses, and pigs, for example.Anti-germinants have the advantage that replicating bacteria will not beunder selective pressure, thus reducing the possibility of resistancedevelopment. Further, since NE is an extracellular, intestinalinfection, compounds need only to be optimized for retention in thegastrointestinal tract.

D. Feed Compositions

Compounds for use as described herein can be incorporated intocompositions for experimental use or for administration to fowl that mayexperience adverse effects (e.g., NE) from exposure to germinated C.perfringens. A composition can include, for example, one or moreheteroaromatic compounds (e.g., 2-MOB or 2-MTB, an analog thereof, or apharmaceutically acceptable salt thereof) as described herein, incombination with a carrier.

Thus, disclosed are feed compositions comprising feed components and acompound having a structure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR6Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino.

Suitable concentrations of a compound within a composition can rangefrom, for example, about 0.1 nM to about 100 mM (e.g., about 0.1 nM toabout 1 nM, about 1 nM to about 10 nM, about 10 nM to about 0.1 mM,about 0.1 mM to about 0.5 mM, about 0.5 mM to about 1 mM, about 1 mM toabout 5 mM, about 5 mM to about 10 mM, about 10 mM to about 25 mM, about25 mM to about 50 mM, about 50 mM to about 75 mM, or about 75 mM toabout 100 mM).

Suitable carriers can include, without limitation, solvents, suspendingagents, stabilizing agents, or any other vehicle for delivering one ormore compounds to a recipient. Suitable carriers typically are nontoxicto the organism being exposed thereto at the dosages and concentrationsemployed. Carriers can be liquid or solid, and can be selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, and other pertinent transport and chemicalproperties, when combined with one or more compounds and any othercomponents of a given composition. Suitable carriers can include, by wayof example and not limitation, water, saline solution, binding agents(e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers(e.g., lactose and other sugars, gelatin, or calcium sulfate),lubricants (e.g., starch, polyethylene glycol, or sodium acetate),disintegrates (e.g., starch or sodium starch glycolate), and wettingagents (e.g., sodium lauryl sulfate). Useful carriers also can includeaqueous pH buffered solutions or liposomes, as well as buffers such asphosphate, citrate, and other organic acids, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins such as serum albumin, gelatin, orimmunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine,monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose or dextrins, chelating agents such as EDTA, sugaralcohols such as mannitol or sorbitol, salt-forming counterions such assodium, and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

Compositions can be formulated by mixing one or more compounds asdescribed herein with one or more carriers, diluents, and/or adjuvants,and optionally other agents that can be incorporated into formulationsto provide improved transfer, delivery, tolerance, and the like.Compositions can be formulated, e.g., in lyophilized formulations,aqueous solutions, dispersions, or solid preparations, such as tablets,dragees, or capsules. Pharmaceutical compositions can include, withoutlimitation, solutions, emulsions, aqueous suspensions, andliposome-containing formulations. These compositions can be generatedfrom a variety of components that include, for example, preformedliquids, self-emulsifying solids and self-emulsifying semisolids.Emulsions are often biphasic systems comprising of two immiscible liquidphases intimately mixed and dispersed with each other; in general,emulsions are either of the water-in-oil (w/o) or oil-in-water (o/w)variety. Emulsion formulations have been widely used for oral deliveryof therapeutics due to their ease of formulation and efficacy ofsolubilization, absorption, and bioavailability.

Since C. perfringens is an extracellular organism found in the gut, thecompounds and compositions provided herein can be optimized forretention in the gastrointestinal tract. Such optimization can include,for example, adding hydrophobic groups to the structure, orencapsulating the compound in gelatin or other appropriate media.

In some embodiments, a compound as described herein can be combined withfeed (e.g., poultry feed), such that the compound is ingested as thebirds eat. Thus, this document provides feed containing (e.g., mixedwith or coated with) a compound that reduces or prevents germination ofC. perfringens, where the compound is a heteroaromatic compound (e.g.,2-MOB or 2-MTB, or an analog thereof) as described herein. Suitablevarieties of feed include those that are commercially available, forexample.

E. Methods of Making the Compounds

In various aspects, the inventions relates to methods of makingcompounds useful to prevent diseases associated with infections causedby C. perfringens. Thus, in one aspect, disclosed are methods of makingcompounds having a structure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a) R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided thatif Q is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(2a)and R^(5b)b, when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein A¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino.

In various aspects, procedures known in the art (e.g., methods describedby Seijas et al. in, for example, Synlett, 2007, 313-316) can be used tosynthesize benzoazole analogs. For example, various alkyl and arylcarboxylic acids can be individually aliquoted into multi-well plates,and each well can be supplemented with o-aminophenol and Lawesson'sreagent. The mixture can be irradiated in a microwave oven, and crudemixtures can be purified in parallel by recrystallization and/or flashchromatography. A similar procedure can be used to obtain 2-substitutedbenzothiazole derivatives from o-aminothiophenol and the same set ofcarboxylic acids.

In various aspects, 2-substituted benzimidazoles can be prepared usingthe procedure of Ryabukhin et al. (e.g., a procedure as described in JOrg Chem, 2007, 72(19):7417-7419) can be used. Briefly, differentaldehydes can be individually aliquoted into multi-well plates. Eachwell can be supplemented with 1,2-diaminobenzene and DMF. TMSC1 can beadded dropwise to the solution, and each well can be sealed and themixtures heated for 4 hours. After cooling, each reaction mixture can beprecipitated with water and recrystallized from an appropriate solvent.

Compounds according to the present disclosure can, for example, beprepared by the several methods outlined below. A practitioner skilledin the art will understand the appropriate use of protecting groups[see: Greene and Wuts, Protective Groups in Organic Synthesis] and thepreparation of known compounds found in the literature using thestandard methods of organic synthesis. There may come from time to timethe need to rearrange the order of the recommended synthetic steps,however this will be apparent to the judgment of a chemist skilled inthe art of organic synthesis. The following examples are provided sothat the invention might be more fully understood, are illustrativeonly, and should not be construed as limiting.

In one aspect, the disclosed compounds comprise the products of thesynthetic methods described herein. In a further aspect, the disclosedcompounds comprise a compound produced by a synthetic method describedherein. In a still further aspect, the invention comprises a feedcomposition comprising an effective amount of the product of thedisclosed methods and a feed component. In an even further aspect, thefeed component is selected from a vegetable protein, a fat-solublevitamin, a water-soluble vitamin, a trace mineral, and a macro mineral.In a still further aspect, the feed component is water.

1. Route I

In one aspect, substituted benzothiazole and benzoxazole analogs can beprepared as shown below.

Compounds are represented in generic form, with substituents as noted incompound descriptions elsewhere herein, wherein R is selected from S andO, and wherein X is halogen. A more specific example is set forth below.

In one aspect, compounds of type 1.6, and similar compounds, can beprepared according to reaction Scheme 1B above. Thus, compounds of type1.5 can be prepared by an alkylation of an appropriate thione, e.g., 1.4as shown above. Appropriate thiones are commercially available orprepared by methods known to one skilled in the art. The alkylation iscarried out in the presence of an appropriate alkyl halide, e.g., 1.5 asshown above, and an appropriate base, e.g., potassium carbonate, in anappropriate solvent, e.g., dimethylformamide. As can be appreciated byone skilled in the art, the above reaction provides an example of ageneralized approach wherein compounds similar in structure to thespecific reactants above (compounds similar to compounds of type 1.1 and1.2), can be substituted in the reaction to provide substitutedbenzothiazole and benzoxazole analogs similar to Formula 1.3.

2. Route II

In one aspect, aryl substituted benzothiazole and benzoxazole analogscan be prepared as shown below.

Compounds are represented in generic form, with substituents as noted incompound descriptions elsewhere herein, wherein R is selected from S andO, and wherein X is halogen. A more specific example is set forth below.

In one aspect, compounds of type 2.3, and similar compounds, can beprepared according to reaction Scheme 2B above. Thus, compounds of type1.6 can be prepared by an arylation of an appropriate thione, e.g., 1.4as shown above. Appropriate thiones are commercially available orprepared by methods known to one skilled in the art. The arylation iscarried out in the presence of an appropriate Grignard reagent, e.g.,2.2 as shown above, and an appropriate oxidizing agent, e.g.,N-chlorosuccinimide (NCS), in an appropriate solvent, e.g., toluene. Ascan be appreciated by one skilled in the art, the above reactionprovides an example of a generalized approach wherein compounds similarin structure to the specific reactants above (compounds similar tocompounds of type 1.1 and 2.1), can be substituted in the reaction toprovide aryl substituted benzothiazole and benzoxazole analogs similarto Formula 2.3.

3. Route III

In one aspect, N-substituted benzothiazol-2-amine analogs can beprepared as shown below.

Compounds are represented in generic form, with substituents as noted incompound descriptions elsewhere herein. A more specific example is setforth below.

In one aspect, compounds of type 3.7, and similar compounds, can beprepared according to reaction Scheme 3B above. Thus, compounds of type3.5 can be prepared by oxidation of an appropriate thioalkyl, e.g., 1.6as shown above. Appropriate thioalkyls are commercially available orprepared by methods known to one skilled in the art. The oxidation iscarried out in the presence of an appropriate oxidant, e.g.,m-chloroperoxybenzoic acid (m-CPBA). Compounds of type 3.7 can beprepared by a displacement reaction of an appropriate sulfonylalkyl,e.g., 3.5 as shown above. The displacement reaction is carried out inthe presence of an appropriate amine, e.g., 3.6 as shown above, in anappropriate solvent, e.g., dimethylformamide, at an appropriatetemperature, e.g., 70° C. As can be appreciated by one skilled in theart, the above reaction provides an example of a generalized approachwherein compounds similar in structure to the specific reactants above(compounds similar to compounds of type 3.1, 3.2, and 3.3), can besubstituted in the reaction to provide N-substitutedbenzothiazol-2-amines similar to Formula 3.4.

F. Methods for Preventing a Disease Caused by Infection by C.Perfringens in a Subject

In one aspect, disclosed are methods for preventing a disease caused byinfection by Clostridium perfringens in a subject, the method comprisingadministering to the subject an effective amount of a compound having astructure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein A¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c),and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino, thereby preventing the disease caused by infection byClostridium perfringens in a subject.

G. Methods for Inhibiting Germination of at Least One C. PerfringensSpore

This document also provides methods for inhibiting germination of C.perfringens spores in the gut of animals (e.g., domesticated or farmedfowl such as, without limitation, chickens, turkeys, ducks, and geese).The administration of such compounds and compositions thus can preventor reduce the likelihood of occurrence of NE in an animal containingintestinal C. perfringens spores, reduce the occurrence of NE in apopulation of animals in which at least some of the animals containintestinal C. perfringens spores, and treat the occurrence of NE in ananimal in which C. perfringens spores have germinated (e.g., to reduceor prevent transmission of the disease to other animals).

Thus, also disclosed are methods for inhibiting germination of at leastone Clostridium perfringens spore, the method comprising contacting thespore with a compound having a structure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar² provided that ifis NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a) andR^(5b), when present, is independently selected from hydrogen and C1-C4alkyl; wherein R⁶, when present, is selected from hydrogen and C1-C4alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino, thereby inhibiting germination of at least one Clostridiumperfringens spore.

The methods can include administering to one or more animal a compoundor composition in an amount effective to reduce or prevent germinationof C. perfringens, as described herein. In various aspects, an effectiveamount can be from about 0.1 nmol to about 100 mmol (e.g., about 0.1nmol to about 1 nmol, about 1 nmol to about 10 nmol, about 10 nmol toabout 0.1 mmol, about 0.1 mmol to about 1 mmol, about 1 mmol to about 5mmol, about 5 mmol to about 10 mmol, about 10 mmol to about 50 mmol, orabout 50 mM to about 100 mmol). In the methods provided herein, thecompound(s), composition(s), and/or feed can be administered any numberof times during the life of the animal, although it is noted thatadministration throughout the life of the animal can be useful. Thus,using feed containing one or more compounds as described herein may beparticularly useful, as the recipient animal would essentiallyself-administer the compound(s) simply by eating.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

H. EXAMPLES

C. perfringens spore germination can produce different and sometimescontradictory results. See, e.g., (Kato et al., J Biosci Bioeng 2009,108(6):477-483; Paredes-Sabja et al., J Bacteriol 2008,190(4):1190-1201; and Paredes-Sabja et al., Appl Envir Microbiol 2009,75(19):6299-6305). As described in the Examples below, the requirementsfor spore germination was analyzed in seven C. perfringens strains.These studies showed that C. perfringens spores can germinate using twodistinct pathways. The first germination pathway (AA) requiresL-alanine/L-phenylalanine as co-germinants. The AA pathway is enhancedby L-arginine and blocked by L-tryptophan. The second germinationpathway (BA) is more promiscuous, and is activated by a number of bilesalts and amino acids (FIG. 1).

As further described herein, analogs of L-tryptophan and indole weretested as inhibitors of C. perfringens spores germinated using eitherthe AA or BA pathway. Tryptophan analogs inhibited C. perfringens sporesfrom germinating through the AA pathway, while indole analogs inhibitedC. perfringens spores from germinating through the BA pathway. Six otherheteroaromatic compounds were found to strongly inhibit both germinationpathways. The strongest inhibitors, 2-methoxybenzothiazole (MOB, IC₅₀=98pM) and 2-methylthiobenzothiazole (2-MTB, IC₅₀=74 pM), were found toinhibit at concentrations approximately ten times lower than otheranalogs.

1. General Experimental Methods

All chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, Mo.).Thioglycollate medium, peptones, yeast extract, raffinose and agar werepurchased from VWR (Radnor, Pa.). C. perfringens strains JGS1936,JGS1473, JGS1882, JGS1521, JGS4104, JGS4151, and JGS 4064 (Barbara etal., Vet Microbiol 2008, 126:377-382) were obtained from Professor J.Glenn Songer (Iowa State University, Ames, Ia.). The identities ofselected C. perfringens spore preparations were confirmed by 16S RNAsequencing.

2. Testing of Growth Conditions on C. Perfringens Sporulation Yields

C. perfringens strains were plated on 2% agar supplemented with 1% yeastextract, 0.1% sodium thioglycollate, 1.5% protease peptone, and 60 mMNa₂HPO₄. Plates were incubated overnight in an anaerobic environment (5%CO₂, 5% Hz, 90% N₂). Single-cell clones were picked and grown for fourhours in either thioglycollate medium or BHI broth. All C. perfringensstrains were then plated on 2% agar supplemented with 1% yeast extract,0.1% sodium thioglycollate, 60 mM Na₂HPO₄, and 1.5% of a peptone source(protease peptone # 1, protease peptone #2, protease peptone #3, orpotato peptone). Media also were supplemented with 0.5% of afilter-sterilized carbon source (glucose, starch, or raffinose). Someplates were supplemented with theobromine to 0.01% final concentration.Plates were incubated for up to 14 days at 37° C. under anaerobicconditions. Sporulation was quantified by microscopy observation ofculture samples stained using the Schaeffer-Fulton method (Hamouda etal., Lett Appl Microbiol 2002, 34:86-90). Under these conditions, sporesare stained green and vegetative cells are stained red. The approximatenumber of green spores and red vegetative cells were counted in at leastthree independent microscopy fields selected at random. A high level ofsporulation was defined as >40% spores. A medium level of sporulationwas defined as 20-40% spores. A low level of sporulation was defined as<20% spores.

3. Purification of C. Perfringens Spores

Each C. perfringens strain was plated under their best sporulationconditions (Table 1). Plates were incubated for 5-10 days at 37° C. inan anaerobic environment. The resulting bacterial lawns were collectedby flooding with ice-cold deionized water. Spores were pelleted bycentrifugation and resuspended in fresh deionized water. After twowashing steps, spores were separated from vegetative and partiallysporulated cells by centrifugation through a 20%-50% HistoDenz gradient.Spore pellets were washed five times with water, resuspended in 0.1%sodium thioglycollate and stored at 4° C. All spore preparations weremore than 95% pure as determined by microscopy observation ofSchaeffer-Fulton stained aliquots.

4. Preparation of Germinant Solution

AGFK mixture (10 mM L-asparagine, 10 mM D-glucose, 10 mM D-fructose, 50mM KCl) was prepared as previously described (Wax and Freese, JBacteriol 1968, 95:433-438). The defined medium employed was describedelsewhere (Ramirez and Abel-Santos, J Bacteriol 2010, 192:418-425).Briefly, a buffer solution was made with 6.6 mM KH₂PO₄, 15 mM NaCl, 59.5mM NaHCO₃, and 35.2 mM Na₂HPO₄. Three solutions were prepared in usingthis buffer as diluent. The first solution contained all salts at 1000×concentrations (final concentration were 10 mg/l MgSO₄.7H₂O, 5 mg/lFeSO₄.7H₂O, 5 mg/l MnCl₂.4H₂O). The second solution contained vitaminsat 10× concentrations (final 132 concentrations were 0.05 mg/l D-biotin,0.1 mg/l p-amino benzoic acid, 0.05 mg/l thiamine hydrochloride, 0.05mg/l pyridoxine, and 1.0 mg/l nicotinic acid). The third solutioncontained all amino acids except cysteine at 10× (final concentrationswere 10 mM for each amino acid). Cysteine was prepared separately as a10× solution in 0.2 N HCl. To prepare the defined medium, differentsolutions were added to buffer at the final concentrations indicated. Insome samples, inosine was added to 1 mM final concentration.

To determine individual germinants, stock (10×) solutions of L-aminoacids, NaHCO₃, KHCO₃, KCl, KBr, NaCl, NaBr, and bile salts wereindividually prepared in deionized sterile water. Combinations of thesesolutions were tested to determine germinants necessary for C.perfringens spore germination. Table 1 below illustrates the source andoptimal sporulation conditions for each C. perfringens strain.

TABLE 1 Strain Source Inoculum Peptone Theobromine 1936 Bovine neonatalenteritis BHI #1 No effect on sporulation 1882 Porcine necroticenteritis BHI #2 Increases sporulation 1473 Chicken normal floraThioglycollate #1 Reduces sporulation 1473 Chicken normal flora BHI #3Increases sporulation 1521 Chicken necrotic enteritis Thioglycollate #1Required for sporulation 4064 Chicken necrotic enteritis BHI #1 Requiredfor sporulation 4104 Turkey necrotic enteritis BHI #3 Required forsporulation 4104 Turkey necrotic enteritis Thioglycollate #3 Requiredfor sporulation 4121 Human gas gangrene Thioglycollate #2 Required forsporulation

I. Requirements for C. Perfringens Spore Sermination

Changes in light diffraction during spore germination were monitored at580 nm (OD₅₈₀) on a Tecan Infinite M200 96-well 144 plate reader (Tecangroup, Mannedorf, Switzerland). C. perfringens spores wereheat-activated at 65° C. for 30 minutes (Desrosier, and Heiligman, FoodRes 1956, 21:54-62). The spore suspension was cooled to room temperatureand monitored for auto-germination for 30 minutes. Germinationexperiments were carried out with spores that did not auto-germinate.After heat activation, spores were resuspended to an OD₅₈₀ of 1 in AGFK,LB broth, or defined medium. Spore germination rates were evaluatedbased on the decrease in OD₅₈₀ at room temperature. After germinantadditions, OD₅₈₀ was measured at 1 minute intervals for 90 minutes.Relative OD₅₈₀ values were derived by dividing each OD₅₈₀ reading by theinitial OD₅₈₀. Experiments were performed in triplicate with at leasttwo different spore preparations. Germination rates were calculated fromthe initial linear region of the germination curves. Standard deviationswere calculated from at least six independent measurements and weretypically below 20%. Germination was confirmed in selected samples bymicroscopy observation of Schaeffer-Fulton stained aliquots.

To determine amino acid co-germinants, C. perfringens spores wereresuspended in germination buffer (0.1 mM sodium phosphate buffer (pH6.5), 50 mM NaHCO₃) to an OD₅₈₀ of 1. Putative germinants were addedindividually or in combinations to a final concentration of 10 mM. Afteraddition of germinants, spore germination was monitored by the decreasein optical density at 580 nm, as above. Germination rates were set to100% for C. perfringens spores germinated in the presence of L-alanineand L-phenylalanine. Relative germination for other germinantcombinations was calculated as the fraction of germination rate comparedto germination with L-alanine/L-phenylalanine.

To determine bile salt co-germinants, C. perfringens spores wereresuspended in potassium phosphate buffer (pH 6.5) supplemented with 5%KHCO₃, and 150 mM KCl. Spore germination was started by addition of 6 mMtaurocholate, and 6 mM individual amino acids. C. perfringens sporeswere also germinated with 6 mM L-alanine and 6 mM individual bile salts.After addition of germinants, spore germination was monitored as above.Germination rates were set to 100% for C. perfringens spores germinatedin the presence of L-alanine and taurocholate. Relative germination forother germinant combinations was calculated as the fraction ofgermination rate compared to germination with L-alanine/taurocholate.

5. Testing for Inhibitors of C. Perfringens Spore Germination

C. perfringens spores were resuspended in sodium phosphate buffer (pH6.5) supplemented with 5% NaHCO₃, and 150 mM NaCl (for the AA pathway)or potassium phosphate buffer (pH 6.5) supplemented with 5% KHCO₃, and150 mM KCl (for the BA pathway). Spore samples were then individuallysupplemented with 6 mM amino acid or 6 mM bile salt analogs. Sporesuspensions were incubated for 15 minutes at room temperature while theOD₅₈₀ was monitored. If no germination was detected, spores weresupplemented with 6 mM L-alanine/6 mM L-phenylalanine (for the AApathway) or 6 mM L-alanine/6 mM taurocholate (for the BA pathway).Germination rates were set to 100% for C. perfringens spores germinatedin the absence of inhibitor. Relative germination for conditions wascalculated as the fraction of germination rate compared to no inhibitor.

6. Effect Of Buffer and pH on C. Perfringens Spore Germination

Individual C. perfringens spore aliquots were individually resuspendedin 0.1 M sodium phosphate buffer (or 0.1 M potassium phosphate buffer)and pH levels were individually adjusted between 5.5 and 8.0.Germination was started by addition of 6 mM L-alanine/6 mML-phenylalanine (for the AA pathway) or 6 mM L-alanine/6 mM taurocholate(for the BA pathway). Spore germination was monitored as above. For theAA pathway, the germination rate was set to 100% for C. perfringensspores germinated at pH 6.5 in sodium phosphate buffer. For the BApathway, the germination rate was set to 100% for C. perfringens sporesgerminated at pH 6.5 in potassium phosphate buffer. The percentage ofgermination for other conditions was calculated as a fraction of therate of germination at pH 6.5.

7. Effect of Cations and Anions on C. Perfringens Spore Germination

C. perfringens spores were individually incubated for five minutes in0.1 M sodium phosphate buffer, pH 6.5 or 0.1 M potassium phosphatebuffer, pH 6.5. Samples were then individually supplemented with 150 mMKCl, KBr, NaCl, NaBr, KHCO₃, or NaHCO₃. Germination was started byaddition of 6 mM L-alanine/6 mM L-phenylalanine (for the AA pathway) or6 mM L-alanine/6 mM taurocholate (for the BA pathway). Spore germinationwas monitored by the decrease in optical density, as above. For the AApathway, the germination rate was set to 100% for C. perfringens sporesgerminated in sodium phosphate buffer without added salts. For the BApathway, the germination rate was set to 100% for C. perfringens sporesgerminated in potassium phosphate buffer without added salts. Thepercentage of germination for other conditions was calculated as afraction of the rate in the absence of added salts.

8. Effect of Sporulation Media on C. Perfringens Spore Germination

Sporulation conditions can affect the germination response of bacterialspores (Hornstra et al., Appl Environ Microbiol 2006, 72:3746-3749; andRamirez-Peralta et al., Appl Environ Microbiol 2011, 78:2689-2697). Totest if C. perfringens spore germination could be modulated bysporulation media, a matrix of conditions for sporulation was createdwith combinations of different liquid media, solid media, carbonsources, peptones, and additives for every C. perfringens strain used inthe study (Table 2).

All C. perfringens strains tested sporulated in solid media, but not inliquid media. However, it was observed that sporulation was dependentupon which liquid media was used for overnight growth prior to platingin agar (Table 2). For strains JG 1936, JG 1882, JG4064, overnightgrowth in BHI was necessary to induce sporulation upon replating in thecorrect solid media. Other strains (JGS 1521, JG4121), requiredovernight growth in liquid thioglycollate medium to induce sporulationin agar. For other strains (JGS 1473, JG41 04), the liquid media usedfor overnight growth changed the preference of solid media required forsporulation.

TABLE 2 Replated from BH1 Replated from Thioglycollate Peptone numberPeptone number Strain #1 #2 #3 #1* #2* #3* #1 #2 #3 #1* #2* #3* 1936 +++− − +++ − − − − − − − − 1882 − ++ − − +++ − − − − − − − 4064 − − − +++ −− − − − − − − 1521 − − − − − − − − − +++ − − 4121 − − − − − − − − − −+++ − 1473 − − +++ − − + ++ − − +++ − − 4104 − − − − − +++ − − − − + +++All C. perfringens strains were plated on 2% agar supplemented with 1%yeast extract, 0.1% sodium thioglycollate, 60 mM Na2HPO4, 0.5%raffinose, and 1.5% of a peptone source. *Plates also were supplementedwith theobromine to 0.01% final concentration; +++, >40% spores; ++,20-40% spores; +, <20% spores; −, no detectable spores.

Glucose, starch, and raffinose were tested as carbon sources forsporulation. Consistent with results described elsewhere, raffinose wasthe preferred carbon source for C. perfringens sporulation (de Jong etal., J Food Protect 2002, 65:1457-1462). In the present studies, glucoseand starch induced poor sporulation under all conditions tested (Sacks,Appl Environ Microbiol 1983, 46:1169-1175).

Peptone sources have been shown to affect the level of sporulation in C.perfringens strains (Hsieh and Labbe J Food Protect 2007, 70:1730-1734).Peptone protease #1 induced sporulation in strains JGS 1936, JGS 1473,JGS4064, and JGS 1521. Peptone protease #2 was able to inducesporulation in strains JGS 1882 and JGS4121. Peptone protease #3 inducedsporulation in JGS1473 and JGS41 04. Potato peptone can induce highlevels of sporulation in some C. perfringens strains (Hsieh and Labbe,supra). In the present studies, however, potato peptone did not inducesporulation in any of the strains tested.

Theobromine can increase the levels of sporulation in C. perfringensstrains, as described elsewhere (de Jong et al., supra). Indeed, strainsJO54104, JO54064, JOS1521, and JO54121 only sporulated robustly whentheobromine was added to solid media. Strains JO51936, JO51882, andJO51473 sporulated in the absence of theobromine. In the presence oftheobromine, sporulation levels for strain JOS 1936 remained unchanged,increased for strain JOS 1882, and decreased for strain JOS1473.

Aside from differentially affecting sporulation levels, theobromine alsoreduced sporulation times in strains JOS1936, JOS1473, and JOS1882. Inthe absence of theobromine, sporulation was not detected until five dayspost-plating and maximum sporulation level was achieved 7-14 dayspost-plating. In the presence of theobromine, spores could be detectedtwo days after plating and maximum sporulation levels were seen 5-7 dayspost-plating.

Contrary to work described elsewhere, C. perfringens spores failed togerminate with AOFK, KCL/L-asparagine, sodium/phosphate, orL-alanine/inosine mixtures (Kato et al., supra; Paredes-Sabja 2008,supra; and Paredes-Sabja 2009, supra). Like other Clostridium species,C. perfringens spores germinated efficiently in defined medium (FIG.2A). C. perfringens spores germinated at the same rate in defined mediumcontaining only amino acids. Henceforth, this germination response isreferred to as the amino acid-only (AA) germination pathway.

9. Effect of Amino Acids on C. Perfringens Spore Germination

To identify which amino acids are required for germination, C.perfringens spores were exposed to mixtures of small (L-Ala and Gly),polar (L-Ser, L-Thr, and L-Cys), hydrophobic (L-Leu, L-Ile, L-Met, andL-Val), aromatic (L-Phe, L-Tyr, and L-Trp), basic (L-Arg, L-Lys, andL-His), acidic (L-Asp and L-Glu), amide (L-Asn and L-Gln), orconstrained (L-Pro) amino acids. None of these solutions alone wassufficient to trigger spore germination. C. perfringens spores were thenresuspended in solutions containing pairs and trios of the above aminoacid groups. C. perfringens spore germination was only observed insolutions containing mixtures of small and aromatic amino acids. FasterC. perfringens spore germination rates were observed when small andaromatic amino acids were supplemented with basic amino acids.

To further narrow the identity of L-amino acid germinants, all possiblecombinations of small, aromatic, and basic amino acids were testedindividually for their effect on C. perfringens spore germination. Forall strains tested, strong C. perfringens spore germination was seen inthe presence of L-alanine/L-phenylalanine (FIG. 2A). L-arginine was notrequired to trigger germination, but increased germination rates by 20%(FIG. 2B). In the L-alanine/L-phenylalanine germination mixture,L-alanine could not be substituted for glycine, even in the presence ofL-arginine. L-tyrosine could substitute L-phenylalanine, but thegermination rate was more than 50% slower. Addition of L-arginine toL-alanine/L-tyrosine-treated spores increased germination rates morethan 2-fold. C. perfringens spores did not respond toL-alanine/L-tryptophan mixtures. In fact, L-tryptophan behaved as aninhibitor of L-alanine/L-phenylalanine-mediated C. perfringens sporegermination (FIG. 2B).

Sterane compounds can modulate the germination response of C. difficileand C. sordellii spores (Liggins et al, J Bacteriol 2011,193:2776-2783). Taurocholate, a known co-germinant of C. difficilespores, was not sufficient to induce germination in C. perfringensspores. On the other hand, combinations of taurocholate and a variety ofamino acids induced strong C. perfringens spore germination. In fact,only six amino acids did not synergize with taurocholate to inducesignificant C. perfringens spore germination (FIG. 3A). Glycocholate,taurochenodeoxycholate, and taurodeoxycholate also induced C.perfringens spore germination in the presence of amino acids. Cholate,chenodeoxycholate, and deoxycholate did not induce or inhibit C.perfringens spore germination in the presence of L-alanine (FIG. 3B).Henceforth, this germination response is referred to as the bilesalt/amino acid (BA) germination pathway.

Because bile salts serve to solubilize dietary fats (Coleman, BiochemSoc Trans 1987, 15:68S-80S), C. perfringens spores were treated witheither SDS or Triton-X-100. Neither detergent was able to trigger C.perfringens spore germination, even in the presence of excess L-alanine.

D-amino acids can inhibit amino acid-mediated spore germination inBacillus species (Yasuda and Tochikubo, Microbiol Immunol 1984,28:197-207). D-alanine and D-arginine failed to inhibit or induce C.perfringens spore germination in the AA germination pathway, butD-phenylalanine and D-tryptophan both inhibited this pathway. Incontrast, all the D-amino acids tested served as co-germinants withtaurocholate in the BA pathway.

10. Optimal Conditions for C. Perfringens Spore Germination

To define the optimal conditions for C. perfringens spore germination,spores were germinated at different pH levels. In the AA pathway,germination was significantly reduced if sodium phosphate buffer wassubstituted with potassium phosphate buffer (FIG. 4A). In contrast, theBA pathway was only active in the presence of potassium ions (FIG. 4B).For both pathways and in all strains, optimal germination occurred atnear neutral to neutral pH. Germination was significantly reduced abovepH 7.5 or below pH 5.5.

Interestingly, addition of KCl, KBr NaCl, or NaBr did not affect the AApathway response in either potassium phosphate or sodium phosphatebuffer (FIG. 4C). Similarly, NaCl and NaBr did not affect the BAgermination pathway when spores were resuspended in potassium phosphateor sodium phosphate buffer. On the other hand, addition of KCl or KBrinduced the BA pathway in spores resuspended in sodium phosphate buffer(FIG. 4D).

Bicarbonate is an essential co-germinant for some Clostridium species,as described elsewhere (Kato et al., supra; and, Ramirez and Abel-Santos2010, supra). In both the AA and BA germination pathways, addition ofpotassium bicarbonate or sodium bicarbonate increased germination ratefor C. perfringens spores resuspended in both potassium and sodiumphosphate buffers (FIGS. 4C and 4D).

Because C. perfringens spores responded to germinants in a mannersimilar to C. sordellii and C. difficile, all spore preparations weretested by germination and growth in litmus milk medium. As expected forC. perfringens, all samples showed stormy clot fermentation (Ericksonand Deibel, Appl Environ Microbiol 1978, 36:567-571). The identities ofselected spore samples were further confirmed by repeating 16S rRNAsequencing.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otheraspects of the invention will be apparent to those skilled in the artfrom consideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

Wwhat is claimed is:
 1. A method for preventing a disease caused byinfection by Clostridium perfringens in a subject, the method comprisingadministering to the subject an effective amount of a compound having astructure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino, thereby preventing the disease caused by infection byClostridium perfringens in a subject.
 2. The method of claim 1, whereinR¹ is selected from hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, Cy¹, andAr².
 3. The method of claim 1, wherein R¹ is C1-C4 alkyl.
 4. The methodof claim 1, wherein each of R^(2a), R^(2b), R^(2c), and R^(2d) ishydrogen.
 5. The method of claim 1, wherein Z is selected from O andNR⁴; wherein R¹ is selected from hydrogen, C1-C3 alkyl, and C1-C3haloalkyl; wherein each of R^(2a), R^(2b), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂,C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 alkylamino, and(C1-C3)(C1-C3) dialkylamino; and wherein R⁴, when present, is selectedfrom hydrogen and C1-C3 alkyl.
 6. The method of claim 1, wherein thecompound has a structure represented by a formula:


7. The method of claim 1, wherein the compound is selected from:


8. The method of claim 1, wherein the disease is necrotizing enteritis.9. The method of claim 1, wherein the subject is a farm animal.
 10. Themethod of claim 9, wherein the farm animal is selected from a chicken, aturkey, a goose, a duck, a cow, a sheep, a horse, and a pig.
 11. Amethod for inhibiting germination of at least one Clostridiumperfringens spore, the method comprising contacting the spore with acompound having a structure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2b), R^(2c), and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino, thereby inhibiting germination of at least one Clostridiumperfringens spore.
 12. The method of claim 11, wherein the spore is inthe gut of an animal.
 13. The method of claim 11, wherein Z is selectedfrom O and NR⁴; wherein R¹ is selected from hydrogen, C1-C3 alkyl, andC1-C3 haloalkyl; wherein each of R^(2a), R^(2a), R^(2c), and R^(2d) isindependently selected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂,C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 alkylamino, and(C1-C3)(C1-C3) dialkylamino; and wherein R⁴, when present, is selectedfrom hydrogen and C1-C3 alkyl.
 14. The method of claim 11, wherein thecompound is selected from:


15. A feed composition comprising a feed component and a compound havinga structure represented by a formula:

wherein Q is selected from O, S, and NR³; wherein R³, when present, isselected from hydrogen and C1-C8 alkyl; wherein Z is selected from O, S,and NR⁴; wherein R⁴, when present, is selected from hydrogen and C1-C8alkyl; wherein R¹ is selected from hydrogen, C1-C8 alkyl, C1-C8haloalkyl, —CR^(5a)R^(5b)(C═O)NHN═CR⁶Ar¹, Cy¹, and Ar², provided that ifQ is NR³ then R¹ is not —CR^(5a)R^(5b)(C═O)R⁵; wherein each of R^(5a)and R^(5b), when present, is independently selected from hydrogen andC1-C4 alkyl; wherein R⁶, when present, is selected from hydrogen andC1-C4 alkyl; wherein Cy¹ is selected from C3-C7 cycloalkyl and C2-C7heterocycloalkyl and substituted with 0, 1, or 2 groups independentlyselected from halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy,C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein Ar¹ is selected from aryl and heteroaryl and substituted with 0,1, or 2 groups independently selected from halogen, —OH, —CN, —NO₂,—NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and(C1-C4)(C1-C4) dialkylamino; wherein Ar² is selected from aryl andheteroaryl and substituted with 0, 1, or 2 groups independently selectedfrom halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; andwherein each of R^(2a), R^(2a), R^(2c), and R^(2d) is independentlyselected from hydrogen, halogen, —OH, —CN, —NO₂, —NH₂, C1-C4 alkyl,C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4)dialkylamino.
 16. The composition of claim 15, wherein the feedcomponent is selected from a vegetable protein, a fat-soluble vitamin, awater soluble vitamin, a trace mineral, and a macro mineral.
 17. Thecomposition of claim 15, wherein the composition is a granule.
 18. Thecomposition of claim 15, wherein the composition is a pellet.
 19. Thecomposition of claim 15, wherein the composition further comprises oneor more of an antibiotic, an arsenical, an antioxidant, an antifungal, aprobiotic, a flavoring agent, a binder, a pigment, a preservative, anemulsifier, and a sweetener.
 20. The composition of claim 15, whereinthe compound is selected from: